Biology subjects

de la Pena, A. H.

Publications and source records attributed to de la Pena, A. H..

4 recordsLinked to original sources

bioRxiv2026

Covalent remodeling of CRBN creates a non-canonical neosubstrate interface with NTAQ1

Molecular glue degrader EM12-FS covalently modifies cereblon (CRBN) His353, enabling selective recruitment of the neosubstrate NTAQ1 to the CRL4CRBN ubiquitin ligase. We determined the cryo-EM structure of the NTAQ1-EM12-FS-CRBN-DDB1 complex, revealing a non-canonical neosubstrate interface created by covalent remodeling of the CRBN sensor loop. Imidazylation repositions His353 to eliminate the steric clash that prevents NTAQ1 engagement by reversible IMiDs, and the engineered interface is stabilized by a distinctive T-shaped C-H/{pi} interaction between sulfated His353 and NTAQ1 Phe126. Biochemical and mutational analyses define the determinants of ternary complex formation and ubiquitination. These findings show that site-specific synthetic modification of CRBN can reprogram induced-proximity pharmacology, expanding specificity beyond the G-loop degron and establishing a framework for covalent engineering of new degrader modalities.

biochemistry↗

bioRxiv2022

Molecular glue CELMoD compounds are allosteric regulators of cereblon conformation

Cereblon (CRBN) is an ubiquitin ligase (E3) adaptor protein co-opted by CRBN E3 Ligase Modulatory Drugs (CELMoD) agents that target therapeutically-relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBNs Thalidomide Binding Domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We therefore performed cryo-EM analyses of the DDB1[~]CRBN apo-complex, and compared these structures with DDB1[~]CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an "open" conformation to the canonical "closed" conformation. Importantly, the neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy, and informing structure-guided design strategies to improve therapeutic efficacy.

molecular biology↗

bioRxiv2019

Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation

The 26S proteasome is essential for protein homeostasis and the regulation of vital cellular processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step reaction of protein degradation, the proteasomes regulatory particle, consisting of the lid and base subcomplexes, undergoes major conformational changes whose origin and control are largely unknown. Investigating the Saccharomyces cerevisiae 26S proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring play critical roles in stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states after a substrate has been engaged. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later steps of substrate processing remain unaffected. Similar defects in the early degradation steps are also observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control conformational transitions of the proteasome. These results provide important insight into the network of interactions that coordinate conformational changes with various stages of proteasomal degradation, and how modulators of conformational equilibria may influence substrate turnover.

biochemistry↗

bioRxiv2018

Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

The 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

molecular biology↗